Arthrobacter ureafaciens

To cleave terminal sialic acid residues, cells were treated with bacterial sialidase (Vibrio cholerae, Sigma) at a concentration of 10 µM for 20 min in PBS. Subsequently, the cells were washed with complete medium and used for downstream analysis. Additionally, viral sialidase (active H1N1; Sino Biological) and bacterial sialidase (Arthrobacter ureafaciens; Roche) were used for pretreatment, as indicated in the figure legends. If not stated otherwise, Vibrio cholerae bacterial sialidase was used.

NEU4 sialidase activity was analyzed with 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4MU-NeuAc; Sigma) as a substrate^{59 (link)}. Reactions were set up in triplicate using 30 μg of total proteins with 50 mM Na citrate/phosphate buffer (pH 3.2), 0.1 mM 4MU-NeuAc, and 6 mg/mL BSA in a final volume of 100 μl, and incubated at 37 °C for 30 min. Reactions were stopped by addition of 1 ml of 0.2 M glycine/NaOH (pH 10.8). The fluorescence associated with the release of 4-MU was measured at the excitation wavelength of 365 nm and an emission wavelength of 445 nm with an ARVO MX plate reader (Perkin–Elmer). The sialidase from Arthrobacter ureafaciens (Roche) was used in the assay to obtain a standard curve.

Briefly, 2F-peracetyl-fucose (2F) is a cell-permeable fluorinated fucose derivative that acts as an inhibitor of fucosyltransferases following its uptake and metabolic transformation into a GDP-fucose mimetic [44 (link),45 (link),46 (link)]. We used the protocols described previously [7 (link)]. For 2F (Sigma-Aldrich, US) treatment, 500 μM of 2F or dimethyl sulfoxide (DMSO, negative control) was added to differentiated PIEs and incubated for 3 days at 37 °C before they were infected with RVC as described above (fresh 2F/DMSO were added daily). Cell viability was examined using Trypan blue before infection to ensure no DMSO-associated toxic or other biological effects on PIEs. The infected PIEs were incubated for 2 days before harvesting. For sialidase treatment, MA104 cells in 96-well plates or PIEs were pre-treated with 10 mU (diluted in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM CaCl₂, and 0.02% NaN₃: TNC buffer) of sialidase/neuraminidase (NA) from Arthrobacter ureafaciens (Roche, US) for 1 h at 37 °C before inoculation. TNC buffer was used as a negative control for sialidase treatment. Following sialidase treatment, the PIEs were infected with RVCs and harvested as described above.